Alkaloids from sponge, scaffolds for the inhibitiion of human immunodeficiency virus (hiv)

ABSTRACT

Anti-viral compounds with low cytotoxicity are identified from screening of products found in Red Sea sponges, including the sponge Stylissa carteri. The identified compounds can be brominated pyrrole-2-aminoimidazole alkaloids and derivatives thereof. Specific examples of identified compounds include oroidin, hymenialdisine, and debromohymenialdisine, as well as derivatives thereof. The compounds also can be useful scaffolds or pharmacores for further chemical modification and derivatization. Selected compounds, particularly oroidin, show selective anti-viral HIV-1 activity coupled with reduced cytotoxicity. The compounds can function as HIV reverse-transcriptase inhibitors, and molecular modeling can be used to confirm inhibition.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 62/068,716 filed Oct. 26, 2014 which is hereby incorporated byreference in its entirety.

BACKGROUND

In 2013, there were an estimated 35.3 million people that had beeninfected and were living with HIV and the number has continued toincrease [1]. HIV includes both HIV-1 and HIV-2. Drugs currently used asanti-HIV-1 therapeutics fall under five categories, including inhibitorsof viral entry, membrane fusion, the reverse transcriptase, theintegrase, and the protease. Highly active anti-retroviral therapy(HAART) combines drugs from at least two of the different classes of theanti-retroviral agents available [2]. Nevertheless, the emergence ofdrug resistant retroviruses is still a major issue associated withcurrent anti-HIV-1 drug therapies [3]. Hence, the effectiveness of HIVtherapy relies on the discovery and approval of novel therapeutics,which are able to combat the various viral replication phases of HIV.

The first anti-retroviral medicine, AZT, was approved in 1987 for thetreatment of HIV-1 [4]. This drug terminates DNA strand elongation bythe addition of an azide group at the C-3 of the nucleoside sugar. Thismodification to the 5-carbon sugar is thought to be inspired by thenucleoside analogs produced by the sponge Tethya crypta, that possess anarabinose sugar instead of the deoxyribose sugar required for DNA strandelongation [5]. By substituting the groups bound to the 2′and 3′ carbonsof the nucleoside sugar, a number of HIV-1 reverse transcriptaseinhibitors had been generated. This example demonstrates the chemicalnovelty provided by natural products, which can lead to therapeuticingenuity. However, the process of finding appropriate natural productscan be difficult.

In the past 15 years of clinical trials, it is believed only five novelnatural product pharmacophores were investigated in clinical trials. Themajority of the 133 naturally derived compounds in clinical trials forthe period of 2008-2013 are derivatives of existing pharmacophores thatare already present in existing human medicines. For this same timeperiod, only 2 of the 133 compounds, both derivatives of the cyclosporinA pharmacophore, were investigated as anti-viral therapeutic candidatesin the treatment of Hepatitis C Virus (HCV) [6]. This reveals that thereis a need for novel anti-viral pharmacophores and their derivatives inclinical trials.

A statistical review by Hu et al. [7] illustrates that bioactivitieswere assigned to only approximately 25% of the marine natural productsreported in the literature from 1985-2012. This does not mean that theremaining 75% do not possess bioactivity; instead it suggests adiscrepancy between the rate of discovery for marine natural productsand the investigation of their associated bioactivities. A further lookinto the type of bioactivities reported revealed that 56% of thebioactive compounds were associated with anti-cancer activity, but only3% with anti-viral activity. Interestingly, in years where a greatervariety of disease targets were screened in order to identifyinhibitors, the proportion of reported bioactivity was the greatest.

This illustrates that there is a need to expand marine natural productscreening efforts beyond the detection of anti-cancer activity in orderto fully realize the potential utility of marine secondary metabolites.

One Red Sea sponge, Stylissa carteri, is known to produce a number ofpharmacologically active brominated pyrrole-2-aminoimidazole alkaloids,which are also produced by sponges from the families Agelasidae,Axindellidae, Hymeniacidonidae [8]. A review of the literature showedthat the chemical repertoire of Stylissa sp. is well characterized withnearly 100 compounds reported. Among these compounds, a dimer of oroidinknown as sceptrin along with its derivatives debromosceptrin,dibromosceptrin, and oxysceptrin have been reported to inhibit HerpesSimplex Virus-1 (HSV-1) and Vesicular Stomatitis Virus (VSV) [9]. Thislends support to the aim of identifying molecules with anti-viralactivity from S. carteri. More specific to anti-retroviral activity,another derivative of oroidin, hymenialdisine (HD), has been usedexperimentally to inhibit Nuclear Factor-kB (NFkB), and subsequentlyinhibit transcription from the long terminal repeat (LTR) of the HumanImmunodeficiency Virus (HIV) in vitro [10].

In a nutshell, while the sponge Stylissa carteri is known to produce anumber of secondary metabolites displaying anti-fouling,anti-inflammatory, and anti-cancer activity, the anti-viral potential ofmetabolites produced by S. carteri has not been extensively explored orreported.

US Patent Publication 2010/0280004 reports that the chemical structuresof latonduine A and esters thereof have been described in the reference,Linington et al. (2003) “Latonduines A and B, New Alkaloids Isolatedfrom the Marine Sponge Stylissa carteri: Structure Elucidation,Synthesis, and Biogenetic Implications” Organic Letters, 5: 2735.

SUMMARY

Embodiments described herein include, for example, a variety of methods,compositions, and kits.

For example, a first aspect is a method comprising: subjecting crudeextracts of Stylissa carteri sponge to solid phase extraction to producea library of fractions; conducting at least one first screening assay ofthe library of fractions for inhibition of HIV and for cytotoxicityincluding selecting at least one fraction for further fractionation;fractionating the at least one fraction further to produce at least oneadditional fraction; conducting at least one second screening assay ofthe at least one additional fraction for inhibition of HIV and forcytotoxicity, including selecting one or more fractions with decreasedcytotoxicity for further identification study; identifying HIV inhibitormolecule candidate(s) based on the inhibition of HIV and decreasedcytotoxicity.

In one embodiment, the library of fractions comprises at least fourfractions.

In one embodiment, the first screening assay is an HIV replicationassay. In another embodiment, the second screening assay is an HIVreplication assay. In another embodiment, the first screening assay andthe second screening assay are HIV replication assays. In anotherembodiment, the first screening assay and the second screening assay areHIV-1 replication assays.

In one embodiment, the fractionating is an HPLC fractionating. Inanother embodiment, the fractionating is an HPLC fractionating whichproduces at least 11 additional fractions.

In one embodiment, the identifying comprises use of LC-MS. In oneembodiment, the identifying comprises determining which molecules appearin multiple fractions. In one embodiment, the identified molecules arefurther tested with a biochemical assay. In one embodiment, theidentified molecules are further tested with a biochemical assay whichis a HIV-1 reverse transcriptase assay. In one embodiment, theidentified molecules exhibit at least a 50% inhibition of HIV-1 at 7 μM.In one embodiment, the identified molecules exhibit at least a 50%inhibition of HIV-1 at 13 μM. In one embodiment, the identifiedmolecules display at least a 50% inhibition of viral replication at 50μM with no associated toxicity. In one embodiment, the identifiedmolecules in a biochemical assay inhibit the activity of the HIV-1reverse transcriptase up to 90% at 25 μM. In one embodiment, theidentified molecule is a pyrrole-imidazole alkaloid compound. In oneembodiment, the identified molecule is a brominatedpyrrole-2-aminoimidazole alkaloid compound. In one embodiment, theidentified molecule is oroidin. In one embodiment, the identifiedmolecule is further shown by molecular modeling to inhibit HIV reversetranscriptase.

A second aspect provides for a method for treating a viral infection ina patient, comprising administering to the patient a therapeuticallyeffective amount of a compound, or a pharmaceutically acceptable salt orsolvate thereof, which is a brominated pyrrole-2-aminoimidazole alkaloidcompound.

In one embodiment, the compound is oroidin or a derivative thereof,hymenialdisine or a derivative thereof, or debromohymenialdisine or aderivative thereof.

In one embodiment, the compound is oroidin or a derivative thereof.

In one embodiment, the patient is infected with an HIV virus. In anotherembodiment, the patient is infected with an HIV-1 virus.

A third aspect provides for a method for treating a viral infection in apatient, comprising administering to the patient a therapeuticallyeffective amount of a compound, or a pharmaceutically acceptable salt orsolvate thereof, which is isolated from a Red Sea sponge.

In one embodiment, the Red Sea sponge is Stylissa carter! sponge.

In one embodiment, the patient is infected with an HIV-1 virus.

In one embodiment, the compound is a brominated pyrrole-2-aminoimidazolealkaloid compound. In another embodiment, the compound is oroidin or aderivative thereof.

Other embodiments include use of the compounds described herein, orderivatives thereof (including oroidin and its derivatives) in themanufacture of a medicament and for use of the medicament in treatingpatients and inhibiting HIV, or more particularly, HIV-1, or HIV reversetranscriptase.

In preferred embodiments, the anti-viral potential of Stylissa carteri,in the family Scopalinidae collected from the Red Sea, was investigated.In preferred embodiments, four Solid Phase Extracts (SPE) obtained fromS. carteri were tested with a well-established cell-based screeningsystem (EASY-HIT, [11]), which evaluates both anti-HIV activity andcytotoxicity. In preferred embodiments, the SPE mixtures were furtherresolved by high performance liquid chromatography (HPLC) fractionationin order to identify compounds with anti-HIV bioactivity from the RedSea sponge S. carteri.

This led to the initial identification of three previously characterizedcompounds, i.e. debromohymenialdisine (DBH), hymenialdisine (HD), andoroidin as anti-retroviral molecule candidates. In preferredembodiments, commercially available purified versions of these moleculeswere re-tested to assess their antiviral potential in greater detail.Specifically, as described more below, DBH and HD exhibit a 50%inhibition of HIV-1 at 7 μM and 13 μM, respectively, but both exhibitedcytotoxicity. Conversely, oroidin displayed a 50% inhibition of viralreplication at 50 μM with no associated toxicity. Additionalexperimentation using a biochemical assay revealed that oroidininhibited the activity of the HIV-1 reverse transcriptase up to 90% at25 μM

In sum, S. carteri is a sponge with wide geographical distribution thathas been shown in preferred embodiments to produce bioactive secondarymetabolites, some of which are also present in members of three othersponge families. The three metabolites detected in this study, DBH, HDand oroidin, were characterized previously many years ago. Thisinvestigation revealed that these compounds are able to inhibit HIV-1replication, and can serve as starting scaffolds for furtherinvestigation.

Moreover, the long time lag between the original discovery of thesecompounds and this discovery of their anti-retroviral activityaccentuates the unevenness of the screening efforts of natural productsand the need for diversification in assay targets. The broaderimplication of this observation is that the anti-viral pharmacophorepotential of many marine natural products might currently be overlooked.Anti-viral screening efforts similar to this one can reveal newmarine-derived agents useful in combating these devastating viraldiseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Three biological replicates of S. carteri SPE F1 were eachtested for the ability to inhibit HIV-1 infection of HIV-1 permissiveLC5-RIC reporter cells in the EASY-HIT assay; (B) Vitality of theinfected LC5-RIC cells treated with three biological replicates of S.carteri SPE F1 and assessed by the MTT assay. The error bars representone standard deviation from the mean. P values between the six testedconcentrations for SPE F1 for the % infection and % viability areP=0.2738 and P=0.0001, respectively.

FIG. 2. (A) HIV infection of RIC cells: Three biological replicates of11 HPLC fractions (approx. 20 μg/ml each) generated from S. carteri SPEF1 were tested for the ability to inhibit HIV-1 replication in theEASY-HIT assay; (B) The vitality of the infected LC5-RIC reporter cellstreated with the three biological replicates of HPLC fraction 1-11generated from S. carteri SPE F1 were assess by the MTT assay. The errorbars represent one standard deviation from the mean. P values betweenthe 11 tested HPLC fractions for the % infection and % viability areP=0.0001 and P=0.0001, respectively.

FIG. 3. (A) Overlay of LC-MS chromatograms for HPLC fractions 2 (black)and 6 (red) generated from S. carteri SPE fraction 1; and (B) a spectrumof the m/z values common to both HPLC fraction 2 and 6 between minutes17-40. The three known compounds 164 debromohymenialdisine (m/z [M+H]+246, C₁₁H₁₂N₅O₂), 10Z-hymenialdisine (m/z [M+H]+ 324, C₁₁H₁₁BrN₅O₂),oroidin (m/z [M+H]+ 389, C₁₁H₁₂Br₂N₅O) have the greatest relativeabundance among the shared compounds common to both HPLC fractions 2 and6.

FIG. 4. (A) The single compounds DBH, HD, and oroidin from S. carteriare tested for the ability to inhibit HIV-1 replication in HIV-1permissible LC5-RIC reporter cells in the EASY-HIT assay; (B) Vitalityof the infected LC5-RIC reporter cells treated with the single compoundsDBH, HD, and oroidin from S. carteri was assessed by the MTT cellproliferation assay. The error bars represent one standard deviationfrom the mean. The P values between the 6 tested concentrations of eachof the three compounds are reported first for the % infection and then %viability graph as follows: debromohymenialdisine, P=0.0001, P=0.0001;hymenialdisine, P=0.0001, P=0.0001; oroidin, P=0.0001, P=0.0001.

FIG. 5. Inhibitory activity of DBH, HD and oroidin from S. carteri onthe HIV-1 RT in a biochemical assay. The error bars represent onestandard deviation from the mean. The P values between the 11 testedHPLC fraction are reported for % activity of the RT as follows:debromohymenialdisine, P=0.0023; hymenialdisine, P=0.169; oroidin,P=0.0001.

FIG. 6. (A) Three biological replicates of S. carteri SPE fraction 1, 2,3, 4 were each tested for the ability to inhibit HIV-1 infection ofHIV-1 permissive LC5-RIC reporter cells in the EASY-HIT assay at sixconcentrations; (B) Vitality of the infected LC5-RIC cells treated withthree biological replicates of S. carteri SPE fractions 1, 2, 3, 4 atsix concentrations and assessed by the MTT assay. Error bars representone standard deviation from the mean. The P values between the sixtested concentrations for each SPE fractions are as follows (reportedfirst for the % infection and then % viability): F1, P=0.2738, p=0.0001;F2, P=0.0001, P=0.0001; F3, P=0.1531, P=0.0001; F4, P=0.0556, P=0.0629.

FIG. 7. LC-MS chromatograms (extracted ion chromatograms) of the m/zions associated with debromohymenialdisine, hymenialdisine, and oroidinfor SPE fraction 1 from S. carteri and comparison to the purchasedstandards. (A) retention time for debromohymenialdisine in the SPEfraction 1 of S. carteri. (B) retention time for thedebromohymenialdisine standard (starred). (C) retention time forhymenialdisine in the SPE fraction 1 of S. carteri. (D) retention timefor the hymenialdisine standard (starred). (E) retention time fororoidin in the SPE fraction 1 of S. carteri. (F) retention time for theoroidin standard (starred).

FIG. 8. MS/MS fragmentation pattern for debromohymenialdisine,hymenialdisine, and oroidin using a collision-induced dissociation of 30eV. (A) Fragmentation pattern of debromohymenialdisine in the SPEfraction 1 of S. carteri. (B) Fragmentation pattern of thedebromohymenialdisine standard. (C) Fragmentation pattern ofhymenialdisine in the SPE fraction 1 of S. carteri. (D) Fragmentationpattern of the hymenialdisine standard. (E) Fragmentation pattern oforoidin in the SPE fraction 1 of S. carteri. (F) Fragmentation patternof the oroidin standard.

DETAILED DESCRIPTION Introduction

U.S. priority provisional application 62/068,716 filed Oct. 26, 2014 ishereby incorporated by reference.

The following PhD thesis is incorporated herein by reference:“Bioprospecting of Red Sea Sponges for Novel Anti-Viral Pharmacophores,”Aubrie Elise O'Rourke, 2015, King Abdullah University of Science andTechnology, Thuwal, Kingdom of Saudi Arabia; including Chapters 1 and 3in particular.

References cited herein are incorporated by reference.

In an overview, a Red Sea sponge library is subjected to High ContentScreening and anti-viral candidate fractions are identified. Then, inthis overview, Stylissa carteri is used in the identification of activecompounds with respect to HIV and HIV-1.

Marine Sponge

One step in the method is subjecting crude extracts of Stylissa carterisponge to solid phase extraction to produce a library of fractions.

Marine and Red Sea sponges, including Stylissa carteri, are generallyknown in the art. The background and selection of the sponge isdescribed more fully in the incorporated by reference PhD thesis“Bioprospecting of Red Sea Sponges for Novel Anti-Viral Pharmacophores,”Aubrie Elise O'Rourke, 2015, King Abdullah University of Science andTechnology, Thuwal, Kingdom of Saudi Arabia.

The particular sponge, such as Stylissa carteri, can be selected out ofa plurality of different sponges based on, among other factors, a highcontent screening approach.

The Red Sea is a unique sampling environment. For example, it exhibitshigh temperature and salinity in opposing gradients. Areas of the RedSea can be genetically distinct from the Indian Ocean, and can providedistinct environmental conditions within the Red Sea. Libraries of RedSea sponges can be established covering 15 or more distinct sponges,covering 15 or more genera, covering two or more classes (includingDemospongiae and Calcarea), including 1-8 replicates per sponge, and insome cases identification by spicules.

Solid Phase Extraction (SPE)

Solid phase extraction (SPE) as a pre-fractionation step is generallyknown in the art and described more below in the working examples. Also,background for SPE and SPE itself are described more in the incorporatedby reference PhD thesis “Bioprospecting of Red Sea Sponges for NovelAnti-Viral Pharmacophores,” Aubrie Elise O'Rourke, 2015, King AbdullahUniversity of Science and Technology, Thuwal, Kingdom of Saudi Arabia;including Chapters 1 and 3 in particular.

The SPE can produce a library of fractions, and the library of fractionscan comprise at least two, at least three, at least four, at least five,or more fractions.

The sponge can be ground in liquid nitrogen and placed in methanol andthen dried to HP2OSS for SPE. Multi-step elution can be carried outusing increasing polarity in water-isopropanol mixtures and then usingmethanol. The eluted materials can be subjected to centrifugalevaporation, and fractions can be redissolved in, for example, methanol.

High Content Screening (HCS) can be carried out. For example, fractionscan be clustered to a reference library based on shared phenotypicprofiles (“fingerprinting”). Pre-fractionated marine extracts can beused. An example of a well-characterized reference library ofpharmaceutically active single molecule compounds is LOPAC1280). Thecompounds can cluster according to a shared mechanism of action. Thiscan provide a prediction for the structure class and/or mechanism ofaction for the compounds of a pre-fractionated sponge mixture.

Screening, Assays

Another step provides for conducting at least one first screening of thelibrary of fractions for inhibition of HIV and for cytotoxicity in afirst assay including selecting at least one fraction for furtherfractionation. In some cases, at least two fractions, or at least threefractions, or at least four fractions, or at least five fractions can beselected for further fractionation. In the meantime, the selectedfractions can be subjected to a first assay which is an HIV replicationassay, or more specifically an HIV-1 replication assay.

Screening and assays are generally known in the art. They are alsodescribed more in the PhD thesis which is incorporated herein byreference: “Bioprospecting of Red Sea Sponges for Novel Anti-ViralPharmacophores,” Aubrie Elise O'Rourke, 2015, King Abdullah Universityof Science and Technology, Thuwal, Kingdom of Saudi Arabia; includingChapters 1 and 3 in particular. See also references cited hereinbelowincluding references 11-12 to Kremb et al.

Cell based assays are generally known in the art including anti-viralassays, HIV replication assays, and HIV-1 replication assays. One wantsto maximize inhibition of the viral target while also minimizing oreliminating cytotoxicity.

The so-called “EASY-HIT” screening technology can be used. This is atechnology that uses LC5-RIC cells, a HIV-1 permissive HeLa-derived cellline with a stable fluorescent reporter gene activated by HIV Tat andRev proteins.

US Patent Publication No. 2003/0181375 describes a cell-based HIV assay.

Fractionation and More Screening

Another step provides for fractionating the at least one fractionfurther to produce at least one additional fraction. Fractionation isgenerally known in the art. In one embodiment, the fractionation is achromatographic based fractionation, such as an HPLC fractionation. Thefractionating can produce, for example, at least 3, or at least 5, or atleast 7, or at least 10, or at least 11 additional fractions.

Also provided for is conducting at least one second screening of the atleast one additional fraction for inhibition of HIV and for cytotoxicityin a second assay, including selecting one or more fractions withdecreased cytotoxicity for further identification study. This secondscreening can be essentially the same as the first screening, describedabove. The second screening also can be targeted for HIV and HIV-1.

In one embodiment, the second assay is an HIV replication assay. Inanother embodiment, the first assay and the second assay are HIVreplication assays, and in particular, can be HIV-1 replication assays.Again, so-called “EASY-HIT” screening technology can be used for thesecond assay.

Identification of Molecules, Further Testing

Another step provides for identifying HIV inhibitor moleculecandidate(s) based on the inhibition of HIV and decreased cytotoxicity.

The identification can be carried out by known methods such aschromatography and/or mass spectral analysis. In one embodiment, theidentifying comprises use of LC-MS (liquid chromatography-massspectroscopy). In one embodiment, the identifying comprises determiningwhich molecules appear in multiple fractions. This can be done byoverlaying traces on each other, for example.

In one embodiment, the identified molecules are further tested with abiochemical assay. In another embodiment, the identified molecules arefurther tested with a biochemical assay which is a HIV-1 reversetranscriptase assay.

If the identified molecules are known, then they can be purchased orprepared by methods known in the art, and subjected to further testingto confirm that the compounds isolated from the sponge are the same.

Inhibition of HIV-1 is one useful test, and a particular concentrationcan be selected for the test. For example, in one embodiment, theidentified molecules exhibit at least a 25%, or at least a 50%, or atleast a 75% inhibition of HIV-1 at 7 μM. In another embodiment, theidentified molecules exhibit at least a 25%, or at least a 50%, or atleast a 75% inhibition of HIV-1 at 13 μM.

In another embodiment, the identified molecules display at least a 25%,or at least a 50%, or at least a 75% inhibition of viral replication at50 μM with no associated toxicity.

In addition to cell testing, biochemical testing or assays also can becarried out for HIV and HIV-1 inhibition. The assay can be done withrespect to activity and inhibition for HIV-1 reverse transcriptase. Forexample, in one embodiment, the identified molecules in a biochemicalassay inhibit the activity of the HIV-1 reverse transcriptase up to 30%,or up to 60%, or up to 90% at 25 μM.

The identified molecule can be a pyrrole-imidazole alkaloid compound. Inone embodiment, the identified molecule is a brominatedpyrrole-2-aminoimidazole alkaloid compound.

In one particular embodiment, the identified molecule is oroidin, andthe 10Z-Hymenialdisine and debromohymenialdisine molecules are excluded(due to higher levels of cytotoxicity). The oroidin molecule or compoundis known in the art. See, for example, U.S. Pat. Nos. 5,834,609;7,098,204; and 7,838,681. It can be purchased commercially and itsactivity can be verified. The oroidin structure is:

Oroidin does not have the seven membered lactam ring as found in10Z-Hymenialdisine and debromohymenialdisine. Rather, it has a centrallinear propenyl group which links together the terminal2-amino-imidazole group and the terminal carboxyamide brominated pyrrolering. The structure of 10Z-Hymenialdisine, having the seven memberlactam ring, is shown:

Aso, the structure of debromohymenialdisine, also having the sevenmember lactam ring, is also shown:

Derivatives and synthetic analogs of identified compounds such asoroidin can be prepared as known in the art. For example, side chains orfunctionalities can be introduced. Modification can be carried out onthe pyrrole ring or on the pyridinium ring, for example. Another examplefor derivatizing is described in, for example, Zidan et al., Mar. Drugs,2014, 12, 940-963. WO 01/041768 describes hymenialdisine and derivativesthereof.

Derivatives can include the pharmaceutically acceptable salts. Acidaddition salts can be formed with organic or inorganic acids. Examplesof acids include acetic, ascorbic, maleic, phosphoric, salicylic, and/ortartric acids.

In addition, molecular modeling can be carried out as known in the artto evaluate and help confirm the mechanism of the anti-viral activity.For example, AutoDock software can be used, and the binding of moleculesidentified herein, such as oroidin, to HIV RT can be modeled. The modesof binding in a binding pocket can be evaluated, and the energy of themodes of binding can be evaluated. Hydrophobic binding pockets can bereviewed. Molecular modeling is also described more in the PhD thesiswhich is incorporated herein by reference: “Bioprospecting of Red SeaSponges for Novel Anti-Viral Pharmacophores,” Aubrie Elise O'Rourke,2015, King Abdullah University of Science and Technology, Thuwal,Kingdom of Saudi Arabia; including Chapters 1 and 3 in particular.

The combination of an assay such as EASY-HIT, in combination with HighContent cytological profiling, as well as molecular modeling can providethe best guidance for discovering new applications of known and unknowncompounds.

Kits can be provided to execute the steps and methods described herein.Instructions can be provided with the kits.

The screening steps and methods described herein can be used to producepharmacologically active compounds which can be used to treat patients,particularly those that have a viral infection, or particularly thosethat have an HIV infection, or particularly those that have an HIV-1infection. The compounds can be formulated as needed with one or moreingredients for its use in such patients as known in the art, so theidentified compounds, or derivatives thereof, can be considered as anactive pharmaceutical ingredient (API).

Methods of Treating Patients

The descriptions provided herein can be used in methods of treatingpatients. One aspect provides for a method for treating a viralinfection in a patient, comprising administering to the patient atherapeutically effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, which is a brominated pyrrole-2-aminoimidazolealkaloid compound.

In one embodiment of this method, the compound is oroidin or aderivative thereof, hymenialdisine or a derivative thereof, ordebromohymenialdisine or a derivative thereof. In one embodiment, thecompound is oroidin or a derivative thereof.

In one embodiment, the patient is infected with an HIV virus, whereas inanother embodiment, the patient is infected with an HIV-1 virus.

Still further, another embodiment provides for a method for treating aviral infection in a patient, comprising administering to the patient atherapeutically effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, which is isolated from a Red Sea sponge. TheRed Sea sponge, in one embodiment, is Stylissa carteri sponge.

In one embodiment, the patient is infected with an HIV virus such as theHIV-1 virus.

In one embodiment, the compound is a brominated pyrrole-2-aminoimidazolealkaloid compound. In another embodiment, the compound is oroidin or aderivative thereof.

The therapeutically effective amount can be optimized and adapted, ofcourse, to many particular uses including regulatory factors, deliverymethods, and a host of other factors. In another embodiment, thetherapeutically effective amount is provided in the form of a solidhaving an amount of active ingredient of at least 1 wt. %. In anotherembodiment, the therapeutically effective amount is provided in the formof a solid having an amount of active ingredient of at least 5 wt. %. Asingle does in some cases provides for 100 to 1,000 mg, or 250 to 750 mgper unit dosage form. Dosage can be, for example, 1 to 4 times per day.

Pharmaceutically acceptable carriers can be used. The carrier can be,for example, solid or liquid depending on the administration form.Administered forms include, for example, parentally, rectally,topically, transdermally, or orally. Injectable routes can be used. Oralroutes can include lozenges, compressed tablets, pills, tablets,capsules, drops, syrups, suspensions, or emulsions. Solid oral dosageforms can be used.

In one embodiment, the identified compound is not in a salt form. Inanother embodiment, the identified compound is in the form of apharmaceutically acceptable salt as known in the art. In one embodiment,the identified compound is not in a solvate form. In another embodiment,the identified compound is in the form of a pharmaceutically acceptablesolvate as known in the art. The term pharmaceutically acceptablesolvate comprises the hydrates and the solvent addition forms thatcompounds described herein can form. Examples of such forms are e.g.hydrates, alcoholates, e.g. methanolates, ethanolates and propanolates,and the like. Particular solvates are the ethanolate, e.g. themonoethanolate.

In general, the identified compound can be in a variety of formsincluding salts, stereoisomeric forms, racemic mixtures, prodrugs,esters, solvates, derivatives, or metabolites thereof.

The term “prodrug” is known in the art and means the pharmacologicallyacceptable derivatives such as, for example, esters, amides andphosphates, such that the resulting in vivo biotransformation product ofthe derivative is the active drug as described herein. Prodrugs of acompound are prepared by modifying functional groups present in thecompound in such a way that the modifications are cleaved, either byroutine manipulation or in vivo, to the parent compound. Prodrugs areoften characterized by excellent aqueous solubility, increasedbioavailability and are readily metabolized into the active inhibitorsin vivo.

The compounds described herein can inhibit the HIV reverse transcriptaseand may also inhibit reverse transcriptases having similarity to HIVreverse transcriptase. Such similarity may be determined using programsknown in the art including BLAST. In one embodiment, the similarity atthe amino acid level is at least 25%, interestingly at least 50%, moreinterestingly at least 75%. In another embodiment, the similarity at theamino acid level at the binding pocket, for the compounds of the presentinvention is at least 75%, in particular at least 90% as compared to HIVreverse transcriptase. Compounds described herein can be tested in otherlentivirusses besides HIV-1, such as, for example, SIV and HIV-2.

Another embodiment provides that the composition consists essentiallyof, or consists of, an isolated and purified form of the identifiedcompound, or a pharmaceutically acceptable salt thereof.

Treatment can comprise use of combinations of APIs including inhibitorsof HIV RT and protease inhibitors. HIV inhibitors are generally knownand categories include NRTIs, NNRTIs, NtRTIs, PIs, and fusioninhibitors. See, for example, US Patent Publication Nos. 2014/0142174and 2007/0249655. Triple combinations can be used. The pharmaceuticaldrugs can be taken simultaneously or in sequence.

Preferred Embodiments and Working Examples

Additional preferred embodiments, including working examples, areprovided below.

1.1. Inhibition of HIV-1 Replication by S. carteri Solid Phase Extract(SPE) Fraction 1

Three biological replicates of S. carteri specimens were collected fromdifferent coral reefs in the Red Sea and were pre-fractionated by SolidPhase Extraction (SPE) and subsequently evaluated for anti-HIV activityand cytotoxicity in the EASY-HIT system. SPE fractionation was performedin order to obtain desalted fractions that were enriched forpharmacologically relevant small organic compounds with differingpolarities. This was achieved by extracting a portion of the spongesample with methanol overnight, then drying the extracted material toHP20SS beads. The beads were loaded onto a flash cartridge and elutedwith 25% IPA/H20 (SPE F1), 50% IPA/H20 (SPE F2), 75% IPA/H20 (SPE F3),100% MeOH (SPE F4). The eluates were concentrated to a pellet, weighed,and re-eluted in methanol for subsequent testing.

When tested on the cell-based HIV-1 replication assay, SPE fraction 1(SPE F1) was observed to display the strongest inhibitory effect withthe least amount of confounding toxicity (FIG. 6). This inhibition by S.carteri SPE fraction 1 (SPE F1) occurs in the early phase of HIV-1replication, which suggests inhibition of events in the virusreplication cycle that include viral entry, fusion, reversetranscriptase, and HIV-1 integration. The HIV-1 permissive LC5-RICreporter cells, when treated with serial dilutions from three biologicalreplicates of S. carteri SPE F1, displayed less than 1-12% infectedcells at concentrations as low as 2.34 μg/ml (FIG. 1a ).

However, these fraction replicates also showed clear effects on themetabolic activity of cells determined by MTT assay (FIG. 1b ), which isoften used as a parameter of cell viability. A caveat in regard tocell-based assays is that cytotoxic compounds will reduce overall cellnumbers and as a result the overall reporter expression will also bereduced. If effects on reporter expression are viewed without cellviability data, this can lead to a false positive hit. Conversely, whenscreening mixtures with a cell-based assay, a mixture which harborseffective compounds can be overlooked during screening and ruled out onaccount of the one or more cytotoxic compounds that mask the activity ofthe effective compound lending to false negatives.

However, the EASY-HIT assay, which has been validated previous to thisstudy [11-13], provides a robust platform to determine whether antiviraland cytotoxic activities of SPE fraction 1 are separable entities byfurther resolving the chemical constituents of the extract mixture andretesting of these fractions. To this end, SPE F1 was subjected tofurther orthogonal HPLC separation steps and the ensuing fractions wereagain tested for the desired anti-retroviral bioactivity.

1.2. Biological Activities of HPLC Fractions Derived from S. carteri SPEFraction 1

The three biological replicates of the parent SPE F1 were furtherfractionated into 11 HPLC fractions using an XDB-C18 column. Thefractions were subsequently tested for the ability to inhibit HIV-1replication in the EASY-HIT assay similar to the SPE fraction. In thisexperimentation, it was found that HPLC fractions 1, 2, and 6 arecapable of inhibiting HIV-1 replication up to 90% (FIG. 2a ); cellvitality testing of the HIV-1 permissive LC5-RIC reporter cells treatedwith HPLC fractions 2 and 6 showed a cell vitality of 50%, but with HPLCfraction 1 it was reduced to 33%. Consequently, only fractions 2 and 6were considered for further studies (FIG. 2b ).

1.3. Identification of HIV-1 Candidate Inhibitors in Bioactive S.carteri HPLC Fractions 2 and 6 by Overlay of Liquid Chromatography-MassSpectrometry (LC-MS) Spectra

In order to identify the compounds responsible for the HIV inhibitoryactivity, HPLC fractions 2 and 6 resulting from SPE Fl were analyzed byLC-MS. The compounds common to the two fractions were assumed to becandidates for anti-viral compounds (FIG. 3a ). The most prominentcompounds in the spectra included: debromohymenialdisine (DBH, m/z[M+H]+ 246, C₁₁H₁₂N₅O₂),), hymenialdisine (HD, 10Z-hymenialdisine (m/z[M+H]+ 324, C₁₁H₁₁BrN₅O₂), and oroidin (m/z [M+H]+ 389, C₁₁H₁₂Br₂N₅O)(FIG. 3b ).

1.4. Three HIV-1 Candidate Inhibitors Hymenialdisine,Debromohymenialdisine, and Oroidin from S. carteri Inhibit HIV-1Replication to Varying Degrees

Oroidin [14], HD and DBH [15,16], isolated and characterized in 1973 andthe 1980s, respectively, have since been synthesized and are availablefor purchase. The single compounds were purchased and evaluated in theEASY-HIT assay for their ability to inhibit HIV-1 replication. The purecompounds were also used as standards and their presence was confirmedin the SPE F1 from the Red Sea S. carteri specimen collected in thisstudy. Tandem mass spectrometry showed a similar retention time andfragmentation pattern for authentic DBH, HD, and oroidin and theircorresponding compound present in the Red Sea S. carteri SPE F1 (FIGS. 7and 8). The DHB treatment inhibited HIV-1 infection down to 30% ofuntreated controls at a concentration of 13 μM without affecting cellvitality. (FIG. 4a, b ). The specific antiviral effect was even strongerfor HD, which clearly inhibited HIV infection to <40% at a concentrationof 3.1 μM without affecting cell vitality. Oroidin showed the weakestantiviral activity, reducing HIV-1 infection to 50% at a concentrationof 50 μM, and proved to be nontoxic for cells at all testedconcentrations (FIG. 4a,b ).

These results support the idea that the SPE F1 contains differentcompounds with anti-viral activities. This includes compounds with bothweak but selective anti-viral activities (e.g. oroidin) and othercompounds that show stronger antiviral activities but may also affectvitality of cells at higher concentrations.

1.5. Oroidin from S. carteri Inhibits the HIV-1 Reverse Transcriptase(RT) in a Biochemical Assay

The procured purified preparations of DBH, HD, and oroidin were screenedin an HIV-1 RT biochemical assay. Interestingly, DBH and HD did not showanti-HIV-1 RT activity. In contrast, oroidin was capable of a 90%inhibition of the HIV-1 RT enzyme at concentrations greater than 25 μM(FIG. 5). Whereas, in the cell-based experimentation presented here,oroidin was shown to reduce HIV-1 replication in a cell-based assay upto 50% when applied at a 50 μM concentration and was non-cytotoxic atall tested concentrations. This suggests that the mechanism ofinhibition of oroidin as an HIV-1 RT inhibitor might be hindered by itspoor ability to be absorbed by a living cell. Thus improvements inoroidin as a prospective scaffold for HIV-1 inhibition would requireadditional medical chemistry modification.

The findings are in contrast to a previous study where oroidin wasobtained as a by-product and was tested for its ability to inhibit HIVreplication. In that study, oroidin was found inactive, but alsonon-cytotoxic. The previous study [17] did not detail the concentrationsused in the assay; however, their negative result may have been due totesting at too low a concentration of oroidin to observe activity in acell-based assay.

Many of the pharmacologically active brominated pyrrole-2-aminoimidazolealkaloids from S. carteri are assumed to be derivatives of the buildingblock oroidin [18]. In this study, oroidin was found to be capable ofinhibiting HIV-1 replication without causing cytotoxicity. HD, aderivative of oroidin, was also found to be capable of inhibiting HIV-1replication; however, cytotoxicity was observed at concentrationsgreater than 3.1 μM. This could be due to the ability of HD to bindNuclear Factor-kB (NFkB), which is crucial for a number of cellularprocesses as well as the ability to induce the process of transcriptionfrom the HIV-1 long terminal repeat (LTR). Similarly, DBH gives rise tocytotoxicity at concentrations greater than 13 μM, a value greater thanthat observed for HD, perhaps on account of HD's lack of theelectronegative bromine atom. Furthermore, HD along with DBH have beenreported to compete with ATP for binding to a number of cyclin-dependentkinases (CDK), including CDK2 [19], where the ability to inhibit CDK2has been shown to decrease the transcription of the HIV-1 virus [18,20].Additionally, DBH and HD have been reported to abrogate theG2-checkpoint of the cell cycle [21]. A G2 arrest is thought to pose areplicative advantage for the virus, as the HIV-1 LTR is most activeduring this phase [22]. This suggests that DBH and HD may also be ableto relieve the cell cycle arrest that supports HIV pathogenesis.However, a prolonged arrest can also lead to apoptosis and result incytotoxicity. NFkB, CDK2, and G2-checkpoint interference are threepossible mechanisms of action to explain the inhibition observed from HDand DBH in this study. However, the inhibition of such ubiquitouscellular proteins and processes could also explain the cytotoxicityassociated with HD and DBH as well as that observed for the parent SPEFl mixture and in HPLC fractions 1, 2 and 6.

From this study, it was observed that oroidin is directly acting toinhibit the retroviral reverse transcriptase, while DBH and HD do notaffect this enzyme. It remains to be determined whether DBH and HDpossess anti-viral activity at other steps of the virus life cycle thatwere not measured in this study. One can assess whether oroidin onlyaffects the reverse transcriptase or whether potential other enzymes orlife cycle steps are affected.

Experimental Section

2.1. S. carteri Specimen Collection and Sample Fractionation

Three biological replicates of S. carteri (described by Denny 1889,identified by Dr. N.J. de Voogd) were collected using gardening sheersfrom three coral reef locations: C26 from Inner Fsar, West (22° 13.974N, 39° 01.760 E), C14 from Inner Fsar, East (22° 13.850 N, 39° 02.216E), and D5 from Al Fahal, East (22° 15.119 N, 38° 57.761 E), using SCUBAat approximately 12 meters depth. The specimens were washed with 1%Phosphate Buffered Saline (PBS) on the boat then wrapped in foil andplaced on ice then stored at −80° C. until processing. Small spongespecimens of 4-10 grams of sponge specimen were ground using a mortarand pestle and then extracted with 15 ml of methanol overnight at 4° C.The following day, the methanol extract was dried onto 150 mg of DiaionHP20SS beads in a CentriVap complete vacuum concentrator (Labconco,Kansas City, USA) on low and the beads were then loaded into a 25 mlFlash Cartridge (Sorbtech, Norcross, Ga.) with 0.795 diameter Frits(Sorbtech, Norcross, Ga.), desalted with deionized water (15 ml, FW1,FW2) and then eluted with 15 ml of each solvent in the following series:25% IPA/H2O (SPE fraction 1), 50% IPA/H2O (SPE fraction 2), 75% IPA/H2O(SPE fraction 3), 100% MeOH (SPE fraction 4) [23]. The solvents werecentrifugally evaporated in a CentriVap complete vacuum concentrator(Labconco, Kansas City, MO) and re-dissolved in 2 ml HPLC grade methanol(Sigma Aldrich) for injection into the HPLC.

2.1.1 Analytical Chemistry of Bioactive S. carteri Fractions

The HPLC fractionation was peak-based and was carried out with a 50 μl(approximately 500 μg of material, dry weight) injection of material anda flow rate of 0.400 ml/min using a ZORBAX Eclipse XDB-C18 LC Column,4.6 mm, 150 mm, 5 μm (Agilent Technologies, Santa Clara, Calif., USA),and the gradient reported in Table 1. SPE fraction 1 and HPLC fractions2 and 6 were analyzed on the Thermo LTQ Orbitrap with 10 μl of materialand a flow rate of 0.800 ml/min using a ZORBAX Eclipse XDB-C18 LCColumn, 4.6 mm, 150 mm, 5 μm and the gradient reported in Table 2.Analysis and prediction of elemental formulas were carried out withXcalibur 2.1 software (Thermo Scientific). MS/MS was performed using aCID of 30 eV on both the parent SPE fraction 1 as well as the threestandards of DBH, HD and oroidin and targeted m/z 246.09, 324.00, and389.93, respectively.

2.1.2 Single Compounds from Bioactive S. carteri Fractions

The individual synthesized compounds debromohymenialdisine were obtainedfrom Enzo Life Sciences, PA, USA (Lot: L20314), 10Z-hymenialdisine (Lot:L27315) and oroidin (Lot: L26427) and product information is as follows:

Debromohymenialdisine

-   Alternative Name: DBH,    4-(2-Amino-4-oxo-2-imidazolin-5-ylidene)-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8-one-   IDENTITY: Identity determined by ¹H-NMR and MS.-   FORMULA: C₁₁H₁₁N₅O₂-   MW: 245.2-   PURITY: 95% (HPLC)-   APPEARANCE: Yellow solid.-   CAS: 75593-17-8, 125118-55-0-   SOURCE/HOST: Isolated from sponge Axinella carteri.-   SOLUBILITY: Soluble in 100% ethanol or DMSO.-   STORAGE: −20° C.-   HANDLING: Keep under inert gas. Protect from light.

Hvmenialdisine

-   IDENTITY: Identity determined by ¹H-NMR and MS-   FORMULA: C₁₁H₁₀BrN₅O₂-   MW: 324.1-   PURITY: 97% (HPLC)-   APPEARANCE: Yellow oil.-   CAS: 82005-12-7-   SOURCE/HOST: Isolated from sponge Axinella carteri.-   SOLUBILITY: Soluble in DMSO (5 mg/ml).-   SHIPPING: AMBIENT-   STORAGE: −20° C.-   HAZARD: TOXIC.-   HANDLING: Protect from light.

Oroidin

-   FORMULA: C₁₁H₁₁Br₂N₅O-   MW: 389.1-   CAS NUMBER: 34649-22-4-   SOURCE/HOST: Isolated from Stylissa sp.-   PURITY: 97% (HPLC, NMR)-   IDENTITY: Identity determined by ¹H-NMR, ¹³C-NMR and MS.-   APPEARANCE: Amorphous solid. (Actual lot: beige solid)-   SOLUBILITY: Soluble in 100% ethanol or DMSO.-   SHIPPING: AMBIENT-   STORAGE: −20° C.-   HANDLING: Protect from light. Keep cool and dry.

The compounds were ordered and screened on the EASY-HIT assay and theHIV-1 RT biochemical assay at molarities below 100 μM in order to assesstheir ability inhibit HIV-1 replication. Statistical significances weredetermined using one-way ANOVA.

2.2. Human Immunodeficiency Virus, Full Virus Screening (EASY-HIT)

The EASY-HIT assay (Kremb et al. [11]) is based on HIV-1 susceptiblereporter cells (LC5-RIC) that contain a stably integrated fluorescentreporter gene that is activated upon successful HIV-1 infection andexpression of the early viral proteins Rev and Tat. LC5-RIC were seededinto 96-well plates (pCLEAR-Plate Black; Greiner Bio-One, Kremsmuenster,Germany) using only the 60 inner wells to avoid variations in theculture conditions in the outer wells. Cells were seeded ata density of10,000 cells per well 24 hours prior to infection. SPE fractions for thebiological replicates C26, C14 and D5, were tested in a serial1:2-dilution with a maximum of 3% methanol or approximately 75 μg/ml forthe S. carteri fraction 1 mixture on the EASY-HIT assay [11]. For theHPLC fractions, SPE fraction 1 HPLC fractions 1-11, were tested on theEASY-HIT assay for biological replicates C26, C14 and D5. The authenticsamples of HD, DBH, and oroidin were screened at 3.1 μM, 6.25 μM, 12.5μM, 25 μM, and 50 μM. All samples were tested in triplicates. Aftercompound addition, LC5-RIC cells were infected by adding 20 μl of HIV-1inoculum (approx. 28.8 ng of p24 for HIV-1LAI derived from HEK 293Tcells) to each well of the plate. Cells were incubated at standard cellculture conditions for 48 hours after infection and were subsequentlyassayed for reporter expression and cell viability.

Reporter expression was determined by measuring the total fluorescentsignal intensity of each culture with a fluorescence microplate reader(Fluoroskan Ascent; ThermoFisher, Schwerte, Germany) at an excitationfilter wavelength of 544 nm and an emission filter wavelength of 590 nmor with a Tecan infinite M200 (Tecan, Crailsheim, Germany) at themonochromator wavelengths 552 nm for excitation and 596 for emission.Statistical significances were determined using one-way ANOVA.

2.2.1. MU Cell Proliferation Assay

The colorimetric assay MU was used in order to assess the viability andactivity of LC5-RIC cells exposed to the unknown sponge mixtures, SPEfraction 1 and HPLC fractions 1-11, for three biological replicates.This cell vitality assay provides a visualization of the process wheremitochondrial enzymes reduce the yellow MTT to purple formazan (ATCC,MTT cell proliferation assay). After reading the HIV reporterexpression, cultures were incubated with 100 μl of MTT solution (0.5 mgof MU; Sigma, Taufkirchen, Germany) in 100 μl of culture medium for 2hours. MTT solution was removed and 100 μl of lysis solution (10%[wt/vol] SDS and 0.6% [vol/vol] acetic acid in dimethyl sulfoxide[DMSO]) was added. The formazan concentrations of the test compounds andthe uninfected control cultures were determined by an ELISA plate reader(Tecan Infinite M200, Tecan Germany GmbH, Crailsheim) and scanned with atest wavelength of 570 nm and a reference wavelength of 630 nm.Statistical significances were determined using one-way ANOVA.

2.3 Human Immunodeficiency Virus, Reverse Transcriptase Biochemical Test

The HIV-1 reverse transcriptase inhibition assay was carried out usingthe commercial kit EnzChek® Reverse Transcriptase Assay (Invitrogen, SanJose, Calif., USA). The reverse transcriptase is a heterodimer with thep66 and the p51 subunits with RNase H activity located in the last 15KDa of the p66 HIV Reverse transcriptase (Calbiochem, Merck-Millipore).Polymerase activity was assessed by its ability to produce RNA-DNAheteroduplexes from a mixture of a long poly (A) template, an oligo dTprimer and dTTP which is then detected by PicoGreen® dsDNA quantitationreagent. The single compound and controls were performed in triplicatedin a 384-well plate format, each with a total reaction mixture of 50 μL.To begin, the poly (A) template was annealed to the oligo dT primer forone hour, then diluted 200 fold in polymerization buffer. Reversetranscriptase (1.2 μl) was added per 300 reactions. This mixture waskept on ice and aliquoted to each well with test compounds. The reactionwas incubated at room temperature for one hour and then 2 μl of 50 nMEDTA was added to stop the reaction. The RNA-DNA heteroduplex waslabeled with PicoGreen and incubated for 5 minutes and then detectedwith the use of a SpectraMax® Paradigm® Multi-mode Microplate DetectionPlatform (Molecular Devices, Sunnyvale, Calif., USA) by scanning at 480nm excitation wavelength and 520 nm emission wavelength. The authenticstandards of HD, DBH, and oroidin were screened at 3.1 μM, 6.25 μM, 12.5μM, 25 μM, 50 μM, and 100 μM. Statistical significances were determinedusing a one-way ANOVA.

REFERENCES AND NOTES

No admission is made that any reference cited herein and/or cited belowas endnotes 1-23 is prior art.

-   1. (UNAIDS). Global report: Unaids report on the global aids    epidemics 2013. JUNPoHA 2013, UNAIDS/JC2502/1/E.-   2. Downs, C. A. et al., Oxidative stress and seasonal coral    bleaching. Free Radical Biology & Medicine 2002, 33, 533-543.-   3. Martinez, J. P. et al., A. Antiviral drug discovery:    Broad-spectrum drugs from nature. Nat Prod Rep 2015, 32, 29-48.-   4. Clercq, D. Curious discoveries in antiviral drug development: The    role of serendipity. Medicinal Research Reviews 2015, 00, No. 0,    1-22, 2015.-   5. Newman, D. J. et al., Therapeutic agents from the sea:    Biodiversity, chemo-evolutionary insight and advances to the end of    Darwin's 200th year. Diving and Hyperbaric Medicine 2009, 39,    216-225.-   6. Butler, M. S., et al., Natural product and natural product    derived drugs in clinical trials. Natural Product Reports 2014, 31,    1612-1661.-   7. Hu, Y. W., et al., Statistical research on the bioactivity of new    marine natural products discovered during the 28 years from 1985    to 2012. Mar Drugs 2015, 13, 202-221.-   8. Yarnold, J. E., et al., High resolution spatial mapping of    brominated pyrrole-2-aminoimidazole alkaloids distributions in the    marine sponge stylissa flabellata via maldi-mass spectrometry    imaging. Mol Biosyst 2012, 401 8, 2249-2259.-   9. Keifer, P. A., et al., Bioactive bromopyrrole metabolites from    the caribbean sponge agelas-conifera. J Org Chem 1991, 56,    2965-2975.-   10. Breton, J. J., et al., The natural product hymenialdisine    inhibits interleukin-8 production in U937 cells by inhibition of    nuclear factor-kappa B. J Pharmacol Exp Ther 1997, 282, 459-466.-   11. Kremb, S., et al., Easy-hit: HIV full-replication technology for    broad discovery of multiple classes of HIV inhibitors. Antimicrobial    Agents and Chemotherapy 2010, 54, 5257-5268.-   12. Kremb, S., et al., Aqueous extracts of the marine brown alga    lobophora variegata inhibit hiv-1 infection at the level of virus    entry into cells. PloS one 2014, 9, e103895.-   13. Helfer, M., et al., The root extract of the medicinal plant    pelargonium sidoides is a potent hiv-1 attachment inhibitor. PloS    one 2014, 9, e87487.-   14. Forenza, S., et al., New bromo-pyrrole derivatives from sponge    agelas-oroides. J Chem Soc Chem Comm 1971, 1129-&.-   15. Sharma, G. M. et al, Characterization of a yellow compound    isolated from the marine sponge phakellia-flabellata. Journal of the    Chemical Society-Chemical Communications 1980, 435-436.-   16. Cimino, G. et al. Isolation and x-ray crystal-structure of a    novel bromo-compound from 2 marine sponges. Tetrahedron Lett 1982,    23, 767-768.-   17. Mohammed, R., et al., Cyclic heptapeptides from the jamaican    sponge stylissa caribica. J Nat Prod 2006, 69, 1739-1744.-   18. Al-Mourabit, A., et al. Biosynthesis, asymmetric synthesis, and    pharmacology, including cellular targets, of the    pyrrole-2-aminoimidazole marine alkaloids. Nat Prod Rep 2011, 28,    1229-1260.-   19. Meijer, L. et al, Inhibition of cyclin-dependent kinases, gsk-3    beta and ck1 by hymenialdisine, a marine sponge constituent. Chem    Biol 2000, 7, 51-63.-   20. Debebe, Z. et al., Iron chelators of the di-2-pyridylketone    thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series    inhibit hiv-1 transcription: Identification of novel cellular    targets-iron, cyclin-dependent kinase (cdk) 2, and cdk9 (vol 79, pg    185, 2011). Mol Pharmacol 2011, 80, 1190-1190.-   21. Curman, D. et al. Inhibition of the g2 DNA damage checkpoint and    of protein kinases chk1 and chk2 by the marine sponge alkaloid    debromohymenialdisine. J Biol Chem 2001, 276, 17914-17919.-   22. Goh, W. C., et al. HIV-1 vpr increases viral expression by    manipulation of the cell cycle: A mechanism for selection of vpr in    vivo. Nat Med 1998, 4, 65-71.-   23. Bugni, T. S. et al., Marine natural product libraries for    high-throughput screening and rapid drug discovery. J Nat Prod 2008,    71, 1095-1098.

TABLE 1 HPLC gradient used to separate the S. carteri SPE fraction 1into HPLC fractions 1-11. Time % H₂0 % Acetonitrile  0 min 65 35  1 min65 35 20 min 30 70 26 min 30 70 28 min 65 35 30 min 65 35

TABLE 2 LC-MS gradient used to characterize the bioactive compounds ofS. carteri SPE fraction 1, HPLC fraction 2 and 6. Time % H₂0 % MeOHFormic acid in each solvent  0 min 90 10 0.10%  5 min 90 10 0.10% 40 min10 90 0.10% 50 min 10 90 0.10% 55 min 90 10 0.10% 60 min 90 10 0.10%

1. An anti-human immunodeficiency virus (HIV) composition obtainedaccording to a method comprising: (i) subjecting crude extracts ofStylissa carteri sponge to solid phase extraction to produce a libraryof fractions; (ii) conducting at least one first screening assay of thelibrary of fractions for inhibition of HIV and for cytotoxicityincluding selecting at least one fraction for further fractionation,(iv) fractionating the at least one fraction further to produce at leastone additional fraction; (v) conducting at least one second screeningassay of the at least one additional fraction for inhibition of HIV andfor cytotoxicity and identifying an HIV inhibitory composition based onthe inhibition of HIV and decreased cytotoxicity; wherein an HIVinhibitory composition inhibits HIV-1 reverse transcriptase.
 2. Thecomposition of claim 1, wherein the library of fractions comprises atleast four fractions.
 3. The composition of any one of claim 1, whereinthe first screening assay is an HIV replication assay and/or, the secondscreening assay is an HIV replication assay.
 4. (canceled)
 5. Thecomposition of claim 3, wherein: (i) the first screening assay and thesecond screening assay are HIV replication assays, optionally, whereinthe HIV is HIV-1; and/or (ii) the fractionating is an HPLCfractionating, and optionally produces at least 11 additional fractions.6-12. (canceled)
 13. The composition of claim 1, wherein the compositionexhibit at least a 50% inhibition of HIV-1 at 7 μM.
 14. (canceled) 15.The composition of claim 1, wherein the composition effectively inhibitsviral replication by at least a at 50 μM with no associated toxicity.16. (canceled)
 17. The composition of claim 1, comprising apyrrole-imidazole alkaloid compound, optionally, wherein the compound isa brominated pyrrole-2-aminoimidazole alkaloid compound.
 18. (canceled)19. The composition of claim 1, comprising oroidin.
 20. (canceled)
 21. Amethod for treating a viral infection in a patient, comprisingadministering to the patient a therapeutically effective amount of thecomposition of claim 1, wherein the method of identifying the compoundfurther comprises identifying HIV inhibitory molecules, and formulatingthe HIV inhibitory molecule with a pharmaceutically acceptable carrier,wherein the composition comprises a brominated pyrrole-2-aminoimidazolealkaloid compound.
 22. The method of claim 21, wherein the compound isoroidin or a derivative thereof, hymenialdisine or a derivative thereof,or debromohymenialdisine or a derivative thereof.
 23. The method ofclaim 22, wherein the compound is oroidin or a derivative thereof. 24.The method of claim 21, wherein the patient is infected with an HIVvirus.
 25. The method of claim 21, wherein the patient is infected withan HIV-1 virus. 26-30. (canceled)
 31. The method of claim 21, whereinthe identified molecules in a biochemical assay inhibits the activity ofthe HIV-1 reverse transcriptase up to 90% at 25 μM.